Methylation and Demethylation of DNA in the Higher Animal

在这个项目中，我的目标是阐明脊椎动物染色体DNA甲基化的调控。为此，我进行了以下实验，得到的结果如下：1。我从鸡（禽）和爪蟾（两栖）中分离出DNA甲基转移酶（MTase） cdna，并确定了核苷酸序列。在培养细胞中表达这两种cdna并分析其性质。过表达小鼠MTase的新生甲基化活性高于鸡MTase。我已经发现了一种新的DNA结合蛋白，MMBP-3，它可以识别甲基化形式的c-Myc结合基序。MMBP-3的DNA结合活性在增殖细胞中较高，在细胞增殖停止时降低。小鼠成肌细胞中MTase的过度表达加速了肌管的形成。这是由在增殖条件下直接诱导MyoD基因转录引起的。MyoD基因的表达与转录起始位点附近特定CpG序列的甲基化呈正相关。我已经分离出一种新的cDNA，命名为AZ1，用去甲基化试剂诱导其表达。AZ1蛋白定位于精母细胞顶体前。

In the project, I aimed to shed light on the regulation of the DNA methylation of chromosomal DNA in vertebrate. For this purpose, I engaged in the following experiments and got the results as follows.1. I have isolated DNA methyltransferase (MTase) cDNAs from chiken (avian) and Xenopus laevis (amphibian) and determined the nucleotide sequences. Both cDNAs were expressed in cultivated cells and their properties were analyzes. Overexpressed mouse MTase showed higher de novo methylation activity than chicken MTase.2. I have identified a novel DNA binding protein, MMBP-3, that specifically recognizes the methylated form of c-Myc binding motif. The DNA binding activity of the MMBP-3 is high in the proliferating cells and decreases when the cell proliferation is arrested.3. Overexpression of mouse MTase in myoblasts accelerates the myotube formation. This is caused by direct induction of MyoD gene transcription under proliferating conditions. The expression of MyoD gene positively correlates with the methylation of specific CpG sequences near transcriptional start site.4. I have isolated a novel cDNA named AZ1, of which expression is induced with demethylating reagent. The AZ1 protein is localized to the pre-acrosome of spermatocytes.